The present invention relates to methods and kits for non-invasive, early detection of cancer and predisposition thereto using CD24, and more particularly, to methods and kits for diagnosing cancer or pre-malignant lesions by determining the expression level of soluble, shedded and/or blebbed CD24 in a biological sample.
Colorectal cancer (CRC) is a major health concern in the Western world as it is the third most common cancer in both men and women in the United States and Israel. This form of cancer develops through a stepwise process that involves a variety of genetic and epigenetic changes that are acquired over several years and eventually culminate in the transformation of normal epithelium into neoplasm. Although the disease has a long latency period, the currently available markers for disease detection are limited to invasive tests such as colon or gastric endoscopy, which often detect the disease while it has already been spread.
Mutations in oncogenes and tumor suppressor genes, abnormal gene expression and genetic defects in a variety of genes are intimately involved in CRC carcinogenesis. On the basis of the allelotypes of a series of colon tumors, Vogelstein and colleagues have showed that the molecular steps that occur after the activation of the APC-β-catenin—Tfc pathway involve a nonlinear accumulation of specific genetic changes that accompany the transition from normal colonic mucosa to metastatic carcinoma. These include mutations in the k-Ras oncogene, changes in methylation patterns, loss of DCC (Deleted in Colorectal Cancer gene) and SMADs [homologs of drosophila Mothers Against Decapentaplegic (MAD) protein, and the C. elegans protein SMA] and mutations in p53.
The currently available screening methods for cancers of the gastrointestinal tract (GI tract) such as colorectal cancer (CRC) include fecal occult blood testing (FOBT). However, although clinical trials have shown that screening with serial FOBT reduces CRC mortality (Mandel J, et al., 1993,), the sensitivity of FOBT is limited [60%; McMahon P M, et al., 2001]. Several markers have been recently suggested as non-invasive diagnostic tools. These include proteins [e.g., fecal calprotectin, lactoferrin, lysozyme, albumin, alpha-1-antitrypsin, carcinoembryonic antigen (CEA), decay-accelerating factor (DAF), minichrosomal maintenance protein (MCM2)] or mRNA (e.g., fecal COX-2) (Kanaoka S., et al., 2004) which can be detected in stool samples, and proteins such as nicotinamide N-methyltransferase (NNMT) (Roessler M, et al., 2005) or proteasome activator complex subunit 3 (PSME3) (Roessler M., et al., 2006), which can be detected in serum samples. However, due to their low sensitivity and specificity, these markers are not in clinical use.
CD24, also known as heat-stable antigen (HSA) in mice, is a heavily glycosilated phosphatidylinositol-anchored mucin-like cell-surface protein. Physiologically, the CD24 protein is expressed mainly on hematopoietic subpopulations of B lymphocytes, various epithelial cells, muscle and neural cells. It plays a crucial role in cell selection and maturation during hematopoiesis and is expressed during the embryonic period, on developing neural and pancreatic cells. In addition, CD24 is a potential ligand for P-selectin which functions as an adhesion molecule that enhances platelets aggregation.
The cellular function of CD24 is still unknown, but recent reports have strengthened its involvement in the initiation of intracellular signal transduction. Schabath et al. (Schabath H, et al., 2006) have associated the expression of CD24 with downregulation in the CXCR-4 chemokine receptor. In addition, CD24 is overexpressed in various malignant tissues including B-cell lymphomas, gliomas, small-cell and non-small cell lung, hepatocellular, renal cell, nasopharyngeal, bladder, uterine, epithelial ovarian, breast, prostate and pancreatic carcinomas (reviewed by Kristiansen et al., 2004). Moreover, its expression was found to correlate with increased growth rate, motility and survival in carcinoma cell lines derived from several organs (Baumann P, et al., 2005; Smith S C, et al., 2006) and with a more aggressive course of cancer. Thus, Weichert W., et al. (2005), found that increased expression of CD24 in the cytoplasm correlates with higher tumor stage, grade and presence of metastasis and concluded that overexpression of CD24 in the cytoplasm (as a result of over production or disturbances in distribution in the cell) is a marker for poorer prognosis. In addition, the role of CD24 in platelet aggregation may explain the involvement with cancer metastases and worse prognosis (Sammar, M., et al., 1994; Aigner, S., et al., 1997; Aigner, S., et al., 1998).
U.S. Pat. Appl. 20040005596 to Li J., et al., discloses methods of diagnosing cancer by determining the level of CD24 in situ in tissue samples suspected to be precancerous or cancerous, thus again necessitating invasional procedures for cancer detection.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of diagnosing cancer, especially at the pre-malignant stage, devoid of the above limitations.